CN109680031A - A kind of extracting method of Elastin peptide - Google Patents
A kind of extracting method of Elastin peptide Download PDFInfo
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- CN109680031A CN109680031A CN201910173879.2A CN201910173879A CN109680031A CN 109680031 A CN109680031 A CN 109680031A CN 201910173879 A CN201910173879 A CN 201910173879A CN 109680031 A CN109680031 A CN 109680031A
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Abstract
The invention discloses a kind of extracting methods of Elastin peptide, belong to bioactive substance extractive technique field.This method are as follows: fish cartilage rejects the impurity such as the flesh of fish, cleans after rubbing, alcohol solution dipping；Tris-HCl buffer, ultrasonication is added；Elastoser, water bath with thermostatic control shaken cultivation is added；Enzymolysis liquid is filtered after enzyme deactivation, abandons filter residue, collects filtrate；Filtrate carries out first time ultrafiltration, collects permeate；Permeate carries out second of ultrafiltration, collects ultrafiltrate；Gained ultrafiltrate, desalination of dialysing, rotary evaporation, the elastin polypeptide after freeze-drying.Extracting method of the invention is simple and quick, and extraction efficiency is high, at low cost.The elastin polypeptide of preparation, reducing power is strong, can effectively clear free radical, and antioxidant effect is good, can significantly improve the content of collagen peptide in skin.It is with a wide range of applications in food, cosmetics, pharmaceuticals industry.
Description
Technical field
The invention belongs to bioactive substance extractive technique fields, and in particular to a kind of extracting method of Elastin peptide.
Background technique
Elastin laminin is one of main composition of elastomer, and elastomer is the elastin laminin and outer bread by homogeneous shape
It is formed around microfibril shell.Microfibril is made of some glycoprotein, for the necessary integrality for keeping elastomer
Ingredient.In the elastic fibrous tissue of body development, glycoprotein microfibril is the skeleton of elastin laminin attachment, often prior to elasticity
Albumen occurs, and is assembled into elastomer for elastin laminin molecule and plays an important role.Elastin laminin is a kind of height intersection
Water-insoluble protein hydrolysate, molecular weight 70KDa.Nonpolar amino acid (hydrophobic amino acid) accounts in elastin laminin molecule
95%, Glycine Levels account for the 1/3 of total amount, and proline accounts for 10%, and hydroxyproline accounts for 1%, with antiacid, alkaline hydrolysis.
Elastin laminin does not dissolve in any solvent in addition to being broken peptide bond agent, is that one kind has rubber ductility and low bullet when there is water
The polymeric material of property modulus.Although to be distributed no collagen extensive for elastin laminin, be also largely present in lung, main artery, certain
In the tissues rich in elasticity such as a little ligaments, skin and auricle.
In photo aging process, Expression of Matrix Metalloproteinases up-regulation causes skin group to cause elastin degradation
Now solar lentigines elastic fibrosis is woven, skin aging is caused.It is used for by adding elastin laminin hydrolysate into cosmetics
Delay skin aging, to anti-light aging, promote skin cell proliferation and induced skin rejuvenation.In addition, elastin laminin is as one
Kind extracellular matrix protein, can form elastomer in conjunction with microfibril in vivo, and major function is passive flexible, imparting
The histoorgan of frequent stress and deformation is with retractility and reversible deformability.Research in recent years discovery, the variation of unisexuality albumen
It is related with a variety of diseases, such as aneurysm, atherosclerosis and face type obstructive disease of lung.
As market is to the growing day by day of Elastin peptide demand, the preparation process of existing Elastin peptide, because preparing work
The problems such as skill is cumbersome, at high cost, low efficiency, with far from meeting needs.Therefore, it is necessary to a kind of more efficient Elastin peptide systems
Standby technique.
Summary of the invention
It is an object of the invention to be directed to the deficiency of existing Elastin peptide extracting method, a kind of Elastin peptide is provided
Extracting method.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of extracting method of Elastin peptide, comprising the following specific steps
(1) fish cartilage rejects the impurity such as the flesh of fish, cleans after rubbing, and 95% alcohol solution dipping 6 hours of 2-3 times of volume are added, go
Ethyl alcohol repeats to repeat to impregnate once with 95% ethanol solution, removes ethyl alcohol, distills water washing 2 times.
(2) Tris-HCl buffer is added in the fish cartilage after washed, then carries out ultrasonication；
(3) in the Cartilage solution after ultrasonication, elastoser, water bath with thermostatic control shaken cultivation is added；
(4) enzyme deactivation filters enzymolysis liquid, abandons filter residue, collects filtrate；
(5) filtrate obtained by step (4) carries out first time ultrafiltration with ultrafiltration membrane, collects permeate under ultra-filtration conditions；
(6) step (5) collects resulting permeate, and the Tris-HCl buffer of the pH 9.0 of 1 times of volume is added, after mixing,
Under ultra-filtration conditions, second of ultrafiltration is carried out, collects ultrafiltrate；
(7) ultrafiltrate obtained by step (6), desalination of dialysing, rotary evaporation, the elastin polypeptide after freeze-drying.
In step (2), the pH of Tris-HCl is 9.0, and additional amount is that 250-300mL Tris- is added in every 100g fish cartilage
HCl buffer；The supersonic frequency of ultrasonic treatment is 60KHz, power 150W, sonication treatment time 30min.
In step (3), the additional amount of elastoser is that 400- is added in Cartilage solution after every 100g ultrasonication
500mg elastoser；Wherein the temperature of water bath with thermostatic control is 30 DEG C, hunting speed 150r/min, and the shaken cultivation time is 1-
1.5h。
In step (4), enzyme deactivation is warming-in-water to 40 DEG C, keeps the temperature 10min.
In step (5), first time ultrafiltration selects the ultrafiltration membrane that molecular cut off is 10 KDa to carry out ultrafiltration, ultra-filtration conditions
It is 0.05-1.0 MPa for operating pressure, operation temperature is 30 DEG C.
In step (6), second of ultrafiltration selects the ultrafiltration membrane that molecular cut off is 1KDa to carry out ultrafiltration, and ultra-filtration conditions are
Operating pressure is 0.05-1.0 MPa, and operation temperature is 30 DEG C.
A kind of Elastin peptide of above method preparation.
A kind of application of the Elastin peptide of above method preparation in skin care item, health care product or drug.
Skin care item, health care product or drug containing above-mentioned unisexuality protein peptides.
The present invention has the advantages that
(1) present invention is raw material using fish cartilage, realizes the devalued efficient utilization of fish.
(2) extracting method of the invention is simple and quick, and extraction efficiency is high, at low cost.
(3) elastin polypeptide prepared by the present invention, reducing power is strong, can effectively clear free radical, and antioxidant effect is good, energy
Enough significantly improve the content of collagen peptide in skin.It is with a wide range of applications in food, cosmetics, pharmaceuticals industry.
Specific embodiment
The present invention is further illustrated by the following examples, but protection scope of the present invention is not limited to following reality
Apply example.
Embodiment 1
A kind of extracting method of Elastin peptide, comprising the following specific steps
(1) 500g fish cartilage rejects the impurity such as the flesh of fish, cleans after rubbing, 95% alcohol solution dipping of 2 times of volumes is added 6 hours,
Ethyl alcohol is removed, repeats to repeat to impregnate once with 95% ethanol solution, removes ethyl alcohol, distill water washing 2 times；
(2) the Tris-HCl buffer of 250mL pH 9.0 is added in the fish cartilage after washed in every 100g, then supersonic frequency
Rate 60KHz, power 150W, sonication treatment time 30min；
(3) 400mg elastoser, 30 DEG C of waters bath with thermostatic control, 150r/ are added in the Cartilage solution after every 100g ultrasonication
Min shaken cultivation 1h；
(4) enzymolysis liquid will be filtered after keeping the temperature 10min enzyme deactivation to 40 DEG C, abandon filter residue, collect filtrate by warming-in-water；
(5) filtrate obtained by step (4) selects the ultrafiltration membrane that molecular cut off is 10 KDa to carry out first time ultrafiltration, ultra-filtration conditions
It is 1.0 MPa for operating pressure, operation temperature is 30 DEG C, collects permeate；
(6) step (5) collects resulting permeate, and the Tris-HCl buffer of 1 times of pH 9.0 is added, and after mixing, selects retention
The ultrafiltration membrane that molecular weight is 1 KDa carries out second of ultrafiltration, and ultra-filtration conditions are that operating pressure is 1.0 MPa, operation temperature 30
℃；
(7) ultrafiltrate obtained by step (6), desalination of dialysing, rotary evaporation, the elastin polypeptide after freeze-drying.
Detect anti-oxidant value (reducing power, the DPPH Scavenging activity, ABTS removing of the present embodiment preparation gained Elastin peptide
Rate), and the influence to model nude mice skin elastin laminin content.
Embodiment 2
A kind of extracting method of Elastin peptide, comprising the following specific steps
(1) 1000g fish cartilage rejects the impurity such as the flesh of fish, cleans after rubbing, 95% alcohol solution dipping 6 that 3 times of volumes are added is small
When, ethyl alcohol is removed, fish cartilage repeats that 95% ethanol solution is used to repeat to impregnate once, removes ethyl alcohol, distill water washing 2 times；
(2) the Tris-HCl buffer of 300mL pH 9.0 is added in the fish cartilage after washed in every 100g, then supersonic frequency
Rate 60KHz, power 150W, sonication treatment time 30min；
(3) 500mg elastoser, 30 DEG C of waters bath with thermostatic control, 150r/ are added in the Cartilage solution after every 100g ultrasonication
Min shaken cultivation 1h；
(4) enzymolysis liquid will be filtered after keeping the temperature 10min enzyme deactivation to 40 DEG C, abandon filter residue, collect filtrate by warming-in-water；
(5) filtrate obtained by step (4) selects the ultrafiltration membrane that molecular cut off is 10 KDa to carry out first time ultrafiltration, ultra-filtration conditions
It is 0.8MPa for operating pressure, operation temperature is 30 DEG C, collects permeate；
(6) step (5) collects resulting permeate, and the Tris-HCl buffer of 1 times of pH 9.0 is added, and after mixing, selects retention
The ultrafiltration membrane that molecular weight is 1 KDa carries out second of ultrafiltration, and ultra-filtration conditions are that operating pressure is 0.8MPa, operation temperature 30
℃；
(7) ultrafiltrate obtained by step (6), desalination of dialysing, rotary evaporation, the elastin polypeptide after freeze-drying.
Detect anti-oxidant value (reducing power, the DPPH Scavenging activity, ABTS removing of the present embodiment preparation gained Elastin peptide
Rate), and the influence to model nude mice skin elastin laminin content.
Embodiment 3
A kind of extracting method of Elastin peptide, comprising the following specific steps
(1) 600g fish cartilage rejects the impurity such as the flesh of fish, cleans after rubbing, 95% alcohol solution dipping 6 that 2.5 times of volumes are added is small
When, ethyl alcohol is removed, fish cartilage repeats that 95% ethanol solution is used to repeat to impregnate once, removes ethyl alcohol, distill water washing 2 times；
(2) the Tris-HCl buffer of 280mL pH 9.0 is added in the fish cartilage after washed in every 100g, then supersonic frequency
Rate 60KHz, power 150W, sonication treatment time 30min；
(3) 450mg elastoser, 30 DEG C of waters bath with thermostatic control, 150r/ are added in the Cartilage solution after every 100g ultrasonication
Min shaken cultivation 1h；
(4) enzymolysis liquid will be filtered after keeping the temperature 10min enzyme deactivation to 40 DEG C, abandon filter residue, collect filtrate by warming-in-water；
(5) filtrate obtained by step (4) selects the ultrafiltration membrane that molecular cut off is 10 KDa to carry out first time ultrafiltration, ultra-filtration conditions
It is 0.5MPa for operating pressure, operation temperature is 30 DEG C, collects permeate；
(6) step (5) collects resulting permeate, and the Tris-HCl buffer of 1 times of pH 9.0 is added, and after mixing, selects retention
The ultrafiltration membrane that molecular weight is 1KDa carries out second of ultrafiltration, and ultra-filtration conditions are that operating pressure is 0.5 MPa, operation temperature 30
℃；
(7) ultrafiltrate obtained by step (6), desalination of dialysing, rotary evaporation, the elastin polypeptide after freeze-drying.
Detect anti-oxidant value (reducing power, the DPPH Scavenging activity, ABTS removing of the present embodiment preparation gained Elastin peptide
Rate), and the influence to model nude mice skin elastin laminin content.
Comparative example
A method of active collagen and elastin laminin being extracted using pigs and cattle cartilage, is included the following steps:
(1) it smashs pig cartilage to pieces slabbing, is then respectively washed 2 times using physiological saline and distilled water, to remove adipose tissue；
(2) acetic acid that molar concentration is 0 .5mol/L is added in through step (1) treated pig cartilage, adjustment pH value is 2
.5, pepsin, 4 DEG C of digestion 72h are then added；
(3) product obtained through step (2) processing is filtered, removes visible impurity；
(4) obtained product will be handled through step (3) and carry out ion exchange, to eliminate the safety of metal ion and nonmetallic ion
And apply upper problem；
(5) concentration of the product obtained through step (4) processing is promoted to 30%；
(6) obtained product will be handled through step (5) carry out TRANSIENT HIGH TEMPERATURE sterilizing, it is micro- by what is generated or bring into preparation process
Biocidal；
(7) obtained product drying-granulating will be handled through step (6), and will obtain active collagen and elastin laminin extract.
Detect anti-oxidant value (reducing power, the DPPH Scavenging activity, ABTS removing of comparative example preparation gained Elastin peptide
Rate), and the influence to model nude mice skin elastin laminin content.
Testing result
1, anti-oxidant value
(1) reducing power measures
The preparation of sample solution: embodiment 1,2,3 and comparative example prepare resulting Elastin peptide sample, with the Tris- of pH7
HCl buffer solution at final concentration of 1mg/mL sample solution.
2mL sample solution be added to 2mL pH7 Tris-HCl buffer is molten and 2 mL 1%(g/L) potassium ferricyanide solution
Mixed liquor in.Then 2mL10%(g/L is added in 50 DEG C of heat preservation 20min in mixture in reaction mixture) trichloroacetic acid,
10min is centrifuged with 3000/min after mixing, takes 2mL supernatant and 2mL distilled water and 0.4mL 0.1%(g/L) ferric chloride solution
It is reacted in reaction tube, its light absorption value at 700nm is measured after 10min.Light absorption value is bigger, and sample reducing power is stronger.
(2) oxygen radical removing ability (DPPH) measures
The configuration of DPPH solution: taking DPPH 1mg to be dissolved in 20mL dehydrated alcohol, ultrasonic 5min, abundant shaken well, takes 1mL should
DPPH solution measures A value at 519nm, and A value is between 1.2-1.3.
The preparation of sample solution: embodiment 1,2,3 and comparative example prepare resulting Elastin peptide sample, with pH7's
Tris-HCl buffer solution at final concentration of 1mg/mL sample solution.
Measurement
A0The measurement of value: taking configured DPPH solution 2mL to be added in test tube, and dehydrated alcohol 1mL is added, is sufficiently mixed,
A value is measured at 519nm.
The measurement of A value: taking configured DPPH solution 2mL to be added in test tube, and 200uL sample solution is added, adds
800uL dehydrated alcohol, mixes well, and after standing in 30min, A value is measured at 519nm.
Inhibiting rate %=(A0-A)/A0×100%
(3) ABTS clearance rate
ABTS stock solution: deionized water dissolves ABTS and potassium peroxydisulfate respectively, make its final concentration be respectively 7.4mmol/L and
The two is uniformly mixed by 2.6mmol/L, is protected from light and is stood 12h.
ABTS working solution: with the diluted ABTS stock solution of pH7 Tris-HCl buffer, make its light absorption value at 734nm
It is 0.70.
The preparation of sample solution: embodiment 1,2,3 and comparative example prepare resulting Elastin peptide sample, with pH7's
Tris-HCl buffer solution at final concentration of 1mg/mL sample solution.
A0The measurement of value: taking configured ABTS working solution 1.8mL to be added in test tube, and the Tris-HCl that pH7 is added is slow
Fliud flushing 0.2mL, is sufficiently mixed, and A value is measured at 25 DEG C of waters bath with thermostatic control 5min, 734nm.
The measurement of A value: taking configured ABTS working solution 1.8mL to be added in test tube, and 0.2mL sample solution is added, then,
It mixes well, A value is measured at 25 DEG C of waters bath with thermostatic control 5min, 734nm.
Inhibiting rate %=(A0-A)/A0×100%
1 embodiment of table and the antioxidant activity for compareing set product
Elastin peptide prepared by the present invention, reducing power, radicals scavenging efficiency are significantly higher than comparative example, anti-oxidant effect
Fruit is good.
2, skin aging model mice efficacy validation
BALBc-nu nude mice is taken, quarantine observation 7 days is normally fed under experimental situation.It is randomly divided into 6 groups, every group 5；Respectively
Are as follows: blank control group, model control group, 1 experimental group of embodiment, 2 experimental group of embodiment, 3 experimental group of embodiment, comparative example experiment
Group.
Skin aging model mice modeling: in addition to blank control group, remaining is organized daily nape and 10%D-gal is subcutaneously injected
1.0g/kg；UV irradiation is carried out simultaneously, illumination wavelength 350-400nm, the daily 40min of irradiation time, it is vertical that light source refuses mouse 40cm
Eminence continues modeling 40 days.Same amount of normal saline, normal lighting conditions raising is subcutaneously injected in the daily nape of blank group.From modeling
Start within 11 days, removes blank control group and model control group, the corresponding embodiment preparation of the daily feeding 100mg/100g of remaining each group
Elastin peptide, continuous feeding 30 days.Detect the content of collagen peptide in skin.
The influence of 2 embodiment of table and comparative example product to collagen content in mouse skin
Elastin peptide prepared by the present invention can effectively promote the content of collagen peptide in ageing skin, can effectively support
Imperial skin aging, the application in anti-aging skin care product have bright prospects.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, is all covered by the present invention.
Claims (8)
1. a kind of extracting method of Elastin peptide, which comprises the following steps:
(1) fish cartilage rejects the impurity such as the flesh of fish, cleans after rubbing, and 95% alcohol solution dipping 6 hours of 2-3 times of volume are added, go
Ethyl alcohol repeats to impregnate once, removes ethyl alcohol, distills water washing 2 times with 95% ethanol solution；
(2) Tris-HCl buffer is added in the fish cartilage after washed, then carries out ultrasonication；
(3) in the Cartilage solution after ultrasonication, elastoser, water bath with thermostatic control shaken cultivation is added；
(4) enzyme deactivation filters enzymolysis liquid, abandons filter residue, collects filtrate；
(5) filtrate obtained by step (4) carries out first time ultrafiltration with ultrafiltration membrane, collects permeate under ultra-filtration conditions；
(6) step (5) collects resulting permeate, and the Tris-HCl buffer of 1 times of volume pH 9.0 is added, after mixing, super
Under the conditions of filter, second of ultrafiltration is carried out, collects ultrafiltrate；
(7) ultrafiltrate obtained by step (6), desalination of dialysing, rotary evaporation obtain elastin polypeptide after freeze-drying.
2. a kind of extracting method of Elastin peptide according to claim 1, it is characterised in that: in step (2), Tris-
The pH of HCl is 9.0, and additional amount is that 250-300mL Tris-HCl buffer is added in every 100g fish cartilage；Ultrasonic treatment
Supersonic frequency is 60KHz, power 150W, sonication treatment time 30min.
3. a kind of extracting method of Elastin peptide according to claim 1, it is characterised in that: in step (3), elastic egg
The additional amount of white enzyme is addition 400-500mg elastoser in the Cartilage solution after every 100g ultrasonication；Wherein constant temperature
The temperature of water-bath is 30 DEG C, hunting speed 150r/min, and the shaken cultivation time is 1-1.5h.
4. a kind of extracting method of Elastin peptide according to claim 1, it is characterised in that: in step (4), enzyme deactivation is
Warming-in-water keeps the temperature 10min to 40 DEG C.
5. a kind of extracting method of Elastin peptide according to claim 1, it is characterised in that: in step (5), for the first time
Ultrafiltration selects the ultrafiltration membrane that molecular cut off is 10 KDa to carry out ultrafiltration；Retention molecule is selected in second of ultrafiltration in step (6)
The ultrafiltration membrane that amount is 1 KDa carries out ultrafiltration.
6. a kind of Elastin peptide prepared such as claim 1-5 the method.
7. application of the Elastin peptide as claimed in claim 6 in skin care item, health care product or drug.
8. a kind of skin care item containing Elastin peptide as claimed in claim 6, health care product or drug.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111171139A (en) * | 2020-02-21 | 2020-05-19 | 海南美肽生物科技有限公司 | Elastin and synthetic method thereof |
| CN112345770A (en) * | 2020-11-06 | 2021-02-09 | 苏州大学附属第一医院 | Application of elastin degradation peptide as vascular calcification marker of peritoneal dialysis patient |
| CN114573682A (en) * | 2022-03-08 | 2022-06-03 | 华南理工大学 | Elastin peptide and preparation method thereof |
| WO2022226731A1 (en) * | 2021-04-26 | 2022-11-03 | 北京盛美诺生物技术有限公司 | Pretreatment method for quantitative detection of undenatured type ii collagen in collagen product or cartilage, and application |
| CN117363681A (en) * | 2023-11-13 | 2024-01-09 | 海南华研胶原科技股份有限公司 | Bonito elastin peptide and preparation method thereof |
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| CN112345770A (en) * | 2020-11-06 | 2021-02-09 | 苏州大学附属第一医院 | Application of elastin degradation peptide as vascular calcification marker of peritoneal dialysis patient |
| WO2022226731A1 (en) * | 2021-04-26 | 2022-11-03 | 北京盛美诺生物技术有限公司 | Pretreatment method for quantitative detection of undenatured type ii collagen in collagen product or cartilage, and application |
| CN114573682A (en) * | 2022-03-08 | 2022-06-03 | 华南理工大学 | Elastin peptide and preparation method thereof |
| CN117363681A (en) * | 2023-11-13 | 2024-01-09 | 海南华研胶原科技股份有限公司 | Bonito elastin peptide and preparation method thereof |
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